A. Field of the Invention
The present invention is directed to a method for the production of recombinant AAV virions containing a gene of interest. More particularly, the present invention is directed to a method for producing rAAV virions free of wild-type AAV and helper virus. The present invention is useful because it produces a highly pure rAAV virion suitable for evaluation of gene therapy protocols and/or use in gene therapy.
B. Background of the Invention
Adeno-associated virus (AAV) is a non-pathogenic, replication-defective parvovirus that has a biphasic life cycle. In the absence of a helper virus, the AAV genome integrates into the hot cell's genome to establish a latent infection. In the presence of a helper virus, such as adenovirus, herpes simplex virus or vaccinia virus, the AAV genome is rescued from latency and is reproduced to establish a lytic infection. See Muzyczka, Curr. Topics Microbiol Immunol. 158 (1992) 97-129; Berns and Linden, BioEssays 17 (1995) 237-245. The helper virus is known to provide the functions needed for AAV replication. In the absence of helper virus, AAV stably integrates into human chromosome 19 site specifically. See Kotin, Proc. Natl. Acad. Sci. 87 (1990) 2211-15; Samulski, EMBO J 10 (1991) 3941-50. The AAV genome consists of a 4.7 kb linear, single-stranded, DNA molecule with 145 bp inverted terminal repeats at each end. The remaining, non-repeated sequences encode for the viral proteins, called rep and cap, involved in virus replication and packaging. The AAV ITRs are the only cis elements required for the viral replication, packaging and integration; the rep and cap functions can be provided in trans. See McLaughlin, J. Virol. 62 (1988) 1963-73; Samulski J. Virol. 63 (1989) 3822-28.
Recombinant AAV (rAAV) vectors are attractive vehicles for human gene therapy because the vectors do not require AAV coding sequences to be expressed viral coding sequences, the viruses (viral particles) is capable of infecting non-dividing and dividing cells efficiently, it has a broad host range and the virions have high physical stability. See Carter, Curr. Opin. Biotech 3 (1992) 533-39; Bachman, Intervirology 11 (1979) 248-54. The most widely used method of generating rAAV particles is called the invention/transfection method. This method involves transfection of host cells, typically 293 cells, with AAV vector plasmid (i.e., plasmid carrying the gene of interest bounded by the AAV ITRs) and with helper plasmid (i.e., plasmid providing the AAV helper functions rep and cap but lacking the ITRs) and infection with adenovirus or herpes virus. See McCown, Brain Res. 713 (1996) 99-107; McLaughlin, J. Virol 62 (1988) 1963-73In this standard method, the helper virus can be separated from AAV vectors by density gradient centrifugation and any residual infectious helper virus inactivated by heat. However, the resulting AAV vector preparations still may contain low levels of infectious helper virus and proteins that may contribute to the immunogenicity of the composition and present a potential hazard for human administration. Also, the helper virus is a pathogenic virus and poses a health risk to laboratory personnel involved in the manufacturing process. Moreover, the large amount of helper virus particles and proteins generated during the infection process makes it difficult to achieve high levels of purity. Heat treatment can inactivate infectious adenovirus, but the treatment leads to a 30-40% drop in the tier of functional rAAV virions and it has been difficult to remove all of the adenoviral proteins, even by multiple rounds of CsCI gradient purifications.
Recently, there have been reports of rAAV production using cell lines providing the necessary helper function for rAAV packaging. See for example, Clark, Gene Therapy 3 (1996) 1124-32; Chiorini, Human Gene Therapy 6 (1995) 1531-42; Clark, Gene Therapy 6 (1995) 132941, Flotte Gene Therapy 2 (1995) 29-37 and Tamayose, Human Gene Therapy 7 (1995) 507-13. this method likewise leads to the generation of infectious adenovirus or herpes virus, which must be purified away from the rAAV particles. Although rAAV particles can be purified on CsCl gradients, often the final preparations are contaminated with adenovirus. Also, CsCl gradient centrifugation protocol is cumbersome to adapt to large-scale manufacturing.
There are several studies employing adenovirus mutants delineating the role of various regions of adenovirus (helper virus) necessary for AAV production. There is evidence to demonstrate that the adenoviral DNA replication genes, E2b, E3 and several adenoviral late genes are not required for AAV replication. See Laughlin, J. Virol. 41 1982) 868-876; Myers, J. Virol. 35 (1980) 65-75; Jay, Proc. Natl. Acad. Sci. 78 (1981) 2927-31; Carter, Virology 126 (1983) 505-16; Ito and Sazuki, J. Gen. Virol. 9 (1970) 243-45; Strauss, J. Virol. 17 (1975) 140-48; and Janik, Virology 168 (1989) 320-29. From these results, it can be predicated that the "accessory" functions that may be necessary to support AAV replication include adenoproteins E2a and E4, as well as VA I RNA. An alternative method of producing rAAV is disclosed in PCT Patent Publication WO 97/17458, published May 15, 1997. In that document, the accessory functions capable of supporting rAAV virion production are provided in the from of one or more vectors containing the adenovirus VA sequence, the adenovirus E4 ORF6 coding region and/or the adenovirus E2a 72 kD coding region. However, upon production of the rAAV virions, numerous adenoviral proteins encoded by the foregoing sequences are produced and must be removed. Moreover, the production of rAAV using the helper vectors described in WO 97/17458 is undesirable because it requires periodic auditing to verify the continued presence and operability of the vectors. Accordingly, there remains a need in the art to provide a system capable of producing commercially significant levels of rAAV virions simply and efficiently.